Antibiotic BU-1880

ABSTRACT

Antibiotic Bu-1880 is a basic peptide of unknown structure which is fermented from a strain of Bacillus circulans, herein designated B255-B6 and deposited in the American Type Culture Collection as A.T.C.C. No. 21828.

United States Patent Kawaguchi et al.

[ Apr. 29, 1975 l l ANTIBIOTIC BU-1880 221 Filed: Dec. 6, 1972 [2l]Appl. No.: 312,451

[52] U.S. Cl 424/119: l95/8() R [51] Int. Cl. H6lk 21/00 [58] Field ofSearch .4 424/119; l95/8() [56] References Cited OTHER PUBLICATIONSMiller The Pfizer Handbook of Microbial Metabolites, McGraw-Hill BookCo., lnc., N.Y., N.Y., 196l, pp. 371 & 372.

Primary E.\uminer lerome D. Goldberg Attorney Agent, or FirmRobert E.Havranek [57 ABSTRACT Antibiotic Bu-l880 is a basic peptide of unknownstructure which is fermented from a strain of Bacillus circ'uluns,herein designated 8255-86 and deposited in the American Type CultureCollection as A.T.CiC. No. 2l 828.

2 Claims, 1 Drawing Figure ANTIBIOTIC BU-1880 BACKGROUND OF THEINVENTION 1. Field of the Invention:

The antibiotic Bu-1880 is a new and novel basic peptide of unknownstructure.

2. Description of the Prior Art:

A. Suzuki, T.; lnouye, K. Fujikawa and Y. Suketa: Studies on thechemical structure of colistin. 1. Fractionation, molecular weightdetermination, amino acid and fatty acid composition. J. Biochem. 54:25-33 (1963).

B. Stansly, P. G.; R. G. Shepard and H. J. White: Polymyxin, a newchemotherapeutic agent. Bull. Johns Hopkins Hospital 81: 43 (1947).

C. Murray, F. J.; P. A. Tetrault, O. W. Kaufmann, H. Koffler, D. H.Peterson and D. R. Collingsworth: Circulin, an antibiotic from anorganism resembling Bacillus circulans. J. Bacteriol. 57: 305 (1949).

D. Vogler, K. and P. O. Studer: The chemistry of the polymyxinantibiotics. Experientia 22: 345-416 (1966).

SUMMARY OF THE INVENTION Antibiotic Bu-1880, a basic peptide of unknownstructure, is fermented from a strain of Bacillus circu- Ians designatedherein as B255-B6 and deposited in the American Type Culture Collectionas A.T.C.C. No. 21828. Chemically the antibiotic is known to containphenylalanine, leucine and oz, 'y-diaminobutyric acid in an approximatemolar ratio of about 1:2:5 and 3- hydroxy-S-methyldecanoic acid. Theantibiotic is active against gram-positive and gram-negative bacteriaboth in vitro and in vivo.

COMPLETE DISCLOSURE This invention relates to a new and novel antibioticcalled Bu-l880. The basic peptide antibiotic has been isolated from thefermentation broth of a new strain of Bacillus circulans, hereindesignated B255-B6, which was isolated from a soil sample collected inIndore, lndia. The producing organism elaborated several activecomponents in the broth, among which the major and the most activecomponent was isolated first, characterized and designated as Bu-l880.Bu-l880 is a basic peptide antibiotic and extractable into n-butanolfrom the fermentation broth. Chemically, it contains phenylalanine,leucine and a, 'y-diaminobutyric acid with an approximate molar ratio of1:2:5, along with 3- hydroxy-8-methyldecanoic acid which is a novelfatty acid moiety never before found as a constituent of an antibiotic.Antibiotic Bu-1880 is active against grampositive and gram-negativebacteria both in vitro and in vivo. Compared with the bacterial peptideantibioticcolistin, Bu-ISSO is more active than colistin againstgram-positive bacteria but less active against gramnegative organisms.Bu-188O is several times less toxic than colistin in terms of the acuteLD The Bu-l880-producing organism, designated strain No. B255-B6 in theBristol-Banyu culture collection, appears to belong to Genus Bacillusand has the characteristics described below:

Morphology Vegetative cell: Rod, 0.5 to 0.7 by 2.5 to 6.0 microns.

Motile. Gram-variable, generally negative.

Spores: Oval to ellipsoidal, 0.8 to 1.0 by 1.2 to 1.5 microns. Centralto terminal. Spore wall thick. Sporangia definitely swollen.

Cultural characteristics 1. Nutrient broth: Pellicle and surface ringformed.

Turbid. Pellicle and sediment viscous.

2. Glucose broth: pH 5.4. Very viscous sediment formed.

3. Colony on nutrient agar: Opaque, convex, entire,

10 later crenated edge, smooth, later becoming wrinkled, viscous.Mediate size, 2 to 5 mm. in diameter. No or scant motile micro-colony.

4. Gelatin-stab: Pellicle and surface ring growth.

5. Milk: pH invariable. Viscous surface ring growth.

Viscous.

6. Growth-temperature: Abundant growth at 28 C. and 42 C. Restrictedgrowth at C. and 45 C. No. growth at 15 C. and 52 C. No growth at pH6.0, 50 C.

20 7. Oxygen demand: Aerobic.

8. NaCl broth: Abundant growth at 2%NaCl. Restricted growth at 3percent-NaCl. No growth at 4%- NaCl.

9. Growth factor: None.

Physiological characteristics 1. Gas production from sugars: Negative 2.Starch hydrolysis: Positive 3. Acetylmethylcarbinol: Negative 4. lndole:Negative 5. Liquefaction of gelatin: Positive 6. Milk: Coagulatedwithout peptonization 7. Reduction of nitrate to nitrite: Positive 8.Utilization of citrate: Negative 9. Utilization of ammonium-salts:Positive 10. Catalase: Positive 1 1. Urease: Negative 40 12. Acidproduction from carbohydrates (with ammonium salt as sole nitrogensource): Glycerol L- Arabinose D-Xylose Rhamnose D-Fructose D-GalactoseD-Glucose D-Mannose Sucrose Lactose Maltose Raffinose -i, lnositol D-Mannitol D-Sorbitol Dulcitol Starch Cellulose lnuline Salicine In viewof the morphological, cultural and physiological characteristicsdescribed above, strain B255-B6 was concluded to be classified to aspecies of Bacillus 0 circulans. The determinative characteristics ofstrain B255-B6 for this taxonomic identification are summarized below.

1. Cell: Rod, Gram-variable. mostly negative.

2. Spores: Oval to ellipsoidal. 3. Sporangia: Definitely swollen. 4. Nogrowth under anaerobic conditions. 5. Catalase: Positive. 6. No gasformation from carbohydrate. 7. Starch: hydrolyzed. 8.Acetylmethylcarbionol: Not produced.

9. lndole: Not produced. 10. No growth at 52 C. l 1. No growth in4%-NaCl broth.

A culture of the living organism has been deposited in the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 andhas been assigned the following name and catalog number:

Antibiotic Production Bacillus circulans strain B255-B6 grows well at28- 42 C. on agar slant. The well-sporulated agar slant was used toinoculate the germination medium having the following composition: 7.5percent soybean meal, 2.0 percent corn starch, 0.33% MgSO -7 H and 1.0%CaCO The seed culture was incubated at 28C. for 2 days on a rotaryshaker (250 rpm), and 2 ml. of the growth was transferred to 100 ml. ofthe fermentation medium (same composition as the seed medium) in aSOO-ml. Erlenmeyer flask.

The progress of the fermentation was followed by the paper disc-agarplate assay using Bacillus subtilis PCI 219 as the test organism. In oneof the shaking flask experiments, the antibiotic production was 250mcg./ml. on the second day (pH 8.0) and reached the maximum of 1000mcg./ml. on the third day (pH 8.5). The pilot tank fermentations gave apeak potency of 100-200 mcg./ml.

Isolation and Purification The fermentation broth (ca. 300L, 100meg/ml.) was extracted at pH 8 with about one-half volume of n-b utanol(15OL). The extract was washed with water, concentrated in vacuo andthen stirred with 5L of N/5 HCl to transfer the activity. The aqueousacidic extract was again backextracted at pH 8 with 10L of n-butanol.The solvent extract was evaporated in vacuo to a small volume and theconcentrate was added with 10 volumes of ethyl acetate-acetone mixture(2:1 to precipitate inactive impurities. The mother liquor wasevaporated in vacuo to give about 60 g. of crude Bu-l 880 as a yellowishbrown solid.

A part of the solid g.) was purified by silica gel chromatography usinga column of silica gel (Wakogel C-200, 3.5 cm. diameter X 70 cm.) whichwas developed by ethanol14 percent ammonia (4:1) solution collecting10-ml. fractions. Tube Nos. 286 through 470 were combined, concentratedin vacuo and then lyophilized to give 3.4 g. of white powder. A traceamount of impurity in the solid was eliminated by repeating the silicagel chromatography.

Physico-chemical Properties Bu-1880 is a white amorphous solid withbasic nature (pKa 8.83 in aqueous ethanol) and melts at 239242 C. withdecomposition. The specific rotation is: [(11 18 (c. 1.0, N/lO HCl). Ithas only end absorption in monomer. The theoretical molecular weight forthis molecule would be 1058.36.

Bu-l88O is soluble in acidic water, aqueous ethanol, aqueous butanol andaqueous dioxane, slightly soluble 5 .in distilled water, methanol,ethanol and n-butanol, and

UV (ultraviolet) regions and shows a peptide-type IR 4 (infrared)spectrum (FIG. 1). The NMR (nuclear magnetic resonance) spectrumindicates a presence of aromatic protons at 87.14 ppm. Analytical dataare:

C, 56.34; H, 8.51; N, 15.92; no halogen, no sulfur. It gives positivereactions with ninhydrin and Ehrlich reagents, and decolorizedpermanganate. The Sakaguchi reaction is negative.

The molecular weight was determined by the ultracentrifugation techniqueand was determined to be in the range of about 1050i50. This wouldappear to support a structure comprised of one part phenylalanine, twoparts leucine, five parts a, 'y-diaminobutyric acid and one part3-hydroxy-S-methyldecanoic acid as a practically insoluble in alkalinewater, acetone and ethyl acetate. The hydrochloride salt is soluble inwater and aqueous methanol.

The paper and thin-layer chromatographic data are as follows:

Paper Chromatography System (ascending) RF n-butanol saturated withwater 035 aqueous 3% NH Cl 0.70 acetone-water (1:1) 0.00n-butanol-methanol-water (4: 1:2) 0.60 above 1.5% methyl orange 0.75benzene-methanol (4:1) 0.00 water 0.00

TLC (Thin Layer TLC plate Chromatography) System RF silica geln-pr0panol-l77r aqueous NH OH 0.10 do. n-butanol-acetic acid water 0.40

(3:1:l) do. n-propanol-pyridine acetic acid- 0.70

water (15:10:32) do. chloroform-pyridine-28% NH OH- 0.85

water (1:4:211) cellulose n-butanol-ethanol-acetic acid- 0.85

water (25:25:3:47)

Bu-1880 was hydrolyzed with 6N HCl in a sealed tube for 16 hours. Theamino acid analysis of the hydrolysate indicated three amino acids,phenylalanine, leucine and a, y-diaminobutyric acid with an approximatemolar ratio of 1:2:5. Extraction of the hydrolysate with ether affordedan acidic substance which was identified with 3-hydroxy-8-methyldecanoicacid.

CH OH Biological Activities Antibiotic preparations used in this sectionwere the hydrochloride salt of Bu-1880 and colistin sulfate. The latterwas a commercial product supplied by Banyu Pharmaceutical Company Ltd.,containing colistins A and B in an approximate ratio of 3:1.

Antibacterial spectrum The minimum inhibitory concentrations (MIC) ofBu-1880 against a wide variety of bacteria were deter mined by theserial agar dilution method on nutrient agar plates of pH 7.0. Theresults are shown in Table 1 along with those of colistin which was usedas a reference antibiotic. Bu-1880 inhibits growth of both grampositiveand gram-negative bacteria including Pseudomonas species. When comparedwith colistin, Bu-l880 is less active than colistin againstgram-negative bacteria but more active against staphylococci and othergram-positive bacilli. Serratia marcescens and Proteus species aremostly resistant to the both antibiotics.

Table I Test Organisms Antibacterial Spectra of Bu-l880 MIC (meg/ml.)

Escherichia do. do. do. Klebsiella do.

do. Pxeudomzmar Serratia do do. Salmonella do. do.

do Shigella do. Shigella Proteus Sarcina M icrocvccus Bacillus do. do

Bu- 1 800 Colistin coli NIl-IJ 3.1 0.4 do. J uhl 3. 1 0.4 do. A20363 3.10.4 do. K 12 3. 1 0.4 pneumoniae D1 1 3.1 0.4 do. A9678 3. 1 0.8 do.A9977 3. 1 0.4 do. A20680 12.5 12.5 aeruginusa D 1 5 6.3 3. 1 do. A992312.5 6.3 do. A9930 3. 1 0.8 do. D1 1 3 1 2.5 6.3 do. Yale 6.3 I .6multophilia A20620 3.1 3.1 sp. A20355 6.3 1.6 do. A20358 12.5 3.1 do.A20368 100 100 marcescens A200 19 1 00 100 do. A20335 100 100 do. A20442100 100 enleriridis A935 1 3. 1 0.8 typhosa Yale 3. 1 0.8 do. NIHJ 3. 10.4 paralyphi A 3.1 0.4 flexneri A9634 3. 1 0.4 dysenteriae 3. 1 0.4.i'onnei Yale 3.1 0.4 t'ulgaris A9436 100 100 do. A9699 6.3 12.5 do.ATCC9920 100 100 morganii A9553 100 100 do. A20455 100 100 do. A9900 6.3100 do. A20454 100 100 aureus Smith 6.3 50 o. No. 193 6.3 50 do. BX-16336.3 25 do. Terajima 3.1 12.5 do. Russell 6.3 50 [men PC] 1001 12.5 50flavus 3. 1 6. 3 mycoides 25 100 cereus ATCC 1 0702 6.3 100 anthracisNo.1 15 6.3 100 Table 2 40 Effect of inoculum size Effect of Media pH onMIC (mcg.lm1.)

Test Organism Bu-1880 Colistin pH6 pI-l7 pI-18 pH6 pI-I7 pI-I8 E. coliNIHJ 6.3 3.1 3.1 0.8 0.8 0.2 Ps. aeruginosa 3.1 3.1 3.1 3.1 3.1 1.6

S. aureus Smith 25 6.3 3.1 100 100 12.5 B. sublilis PCI 1.6 1.6 0.4 253.1 0.2

Effect of inoculum Size (cell/ml.) on MIC (mcg./m1.)

Table 3 The effect of inoculum size of test organisms on the MIC of Bu-1 880 was tested in Nutrient Broth at pH 7.0, the organisms beinginoculated at 10 10 and 10 cell/ml. of the medium. As shown in Table 3,increase of the inoculum size resulted in greated MIC value of Bu-l880in a like fashion as with colistin.

Test Organism E. coli NIHJ 25 3.1 3.1 1.6 0.2

Ps. aeruginosa 12.5 3.1 1.6 12.5 0.4

S. aureus Smith 6.3 3.1 1.6 50 6.3

B. subtilis PCI 1.6 0.8 0.2 3.1 0.2

Effect of serum Increasing concentrations of human serum were added toNutrient Broth to give final serum concentrations of 25 percent, 50percent and 75 percent. The MICs were determined against four testorganisms and colistin was used as a reference. The results are shown inTable 4.

Table 4 Effect of Serum on MIC (meg/ml.)

Bu-l880 Test Organism Colistin E. culi NIHJ Ps. aeruginosa S. aureusSmith B. sublilis PCI Gun but

Table In vivo Activity Test Organism CD (mg/kg.)

Bu-1880 Colistin E. coli .luhl 165 3.4 Ps. ueruginosa D 100 15 S. aureusSmith 80 No protection at non-toxic dose mg./kg.).

Toxicity The acute toxicity of Bu-1880 (hydrochloride salt) wasdetermined in mice. The subcutaneous and intravenous LD were 300 mg./kg.and 37 mg./kg., respectively. In a comparative test, colistinhydrochloride showed the subcutaneous and intravenous LD of 56 mg./kg.and 11 mg./kg., respectively.

Antibiotic Bu-l880 resembles to colistins", polymyxins and circulins inthat they are all bacterial peptide antibiotics containing in thestructure a fatty acid moiety and 5 to 6 moles of a, 'y-diaminobutyricacid I'Iowever, Bu-l880 is different from the others in the. lack ofthreonine in the molecule and the presence of new fatty acid moiety,3-hydroxy-8-methyldecanoic acid, which to our knowledge is the firstoccurrence innatural products.

The antibacterial spectra of the bacterial peptide antibiotics aregenerally limited to either gram-negative or gram-positive. Thecolistin-polymyxin-circulin group of antibiotics are mainly activeagainst gramnegative bacteria, and gramicidin-bacitracin groups areprimarily active against gram-positive organisms. Bu- 1880 is thereforedifferent from these known antibiotics in its broader antibacterialspectrum.

the bowel. Both aerobic and anaerobic flora which are suseptible toBu-1880 is reduced in the large intestine. When accompanied by adequatemechanical cleansing, they are useful in preparing for colonic surgery.

Bu-l880 is effective in the treatment of systemic bacterial infectionswhen administered parenterally in the dosage range of about 250 mg. toabout 3000 mg. per day in divided doses three or four times a day.Generally it is effective when administered at a dosage of about 5.0 to7.5 mg./kg. of body weight every 12 hours.

A preferred embodiment of the present invention is the process for thepreparation of the antibiotic Bu- 1880, a basic peptide comprised ofphenylalanine, leucine and a, y-diaminobutyric acid in an approximatemolar ratio of 1:2:5 and 3-hydroxy-8-methyldecanoic acid, having theinfrared spectrum of FIG. 1 and the following physical characteristics:pKa of 8.83 in aqueous ethanol, m.p. of 239-242 C. with decomposition,[a] =18(c. 1.0, N/lO HCl) and an elemental analysis of C, 56.34; H,8.51; N, 15.92, and a molecular weight of about 1050:50; which processcomprises aerobically fermenting Bacillus circulans A.T.C.C. 21828 andrecovering the antibiotic by extraction.

A most preferred embodiment is the antibiotic Bu- 1880, a basic peptidecomprised of phenylalanine, leucine and a, 'y-diaminobutyric acid in anapproximate molar ratio of 1:225 and 3-hydroxy-8-methyldecanoic acid,having the infrared spectrum of FIG. 1 and the following physicalcharacteristics: pKa of 8.83 in aqueous ethanol, m.p. of 239242 C. withdecomposition, [a] '-18 (c. 1.0, N/lO I-ICl), an elemental analysis ofC, 56.34; H, 8.51; N, 15.92; and a molecular weight of l050i50.

We claim:

1. The antibiotic Bu-1880, a basic peptide comprised of phenylalanine,leucine, a, 'y-diaminobutyric acid and 3-hydroxy-8-methyldecanoic acidin an approximate molar ratio of l:2:5:1, having the infrared spectrumof FIG. 1 and the following physical characteristics: pKa of 8.83 inaqueous ethanol, m.p. of 239-242 C. with decomposition, [a] =18(c. 1.0,N/lO I-lCl), an elemental analysis of C, 56.34; H, 8.51; N, 15.92, and amolecular weight of about 1050fi0.

2. The process for the preparation of the antibiotic Bu-1880, a basicpeptide comprised of phenylalanine, leucine, a, 'y-diaminobutyric acidand 3-hydroxy-8- methyldecanoic acid in an approximate molar ratio of1:2:5: l having the infrared spectrum of FIG. 1 and the followingphysical characteristics: pKa of 8.83 in aqueous ethanol, m.p. of 239242C. with decomposition, [a] =l8 (c. 1.0, N/lO HCl), an elemental analysiscally fermenting Bacillus circulans A.T.C.C. 21828 in acarbohydrate-proteinrich media for a period of about two to about threedays, at a pH of about 8 to about 8.5, at a temperature of about 28 C.,and then recoverof 56.34; -9 and a m l l r gh 5 ing the antibiotic byextraction with n-butanol.

of about 1050-50; which process comprises aerobi-

1. THE ANTIBIOTIC BU- 1880, A BASIC PEPTIDE COMPRISED OF PHENYLALANINE,LEUCINE, A, Y-DIAMINOBUTYRIC ACID AND 3HYDROXY-8-METHYLDECANOIC ACID INAN APPROXIMATE MOLAR RATIO OF 1:2:5:1, HAVING THE INFRARED SPECTRUM OFFIG. 1 AND THE FOLLOWING PHYSICAL CHARACTERISTICS: PKA OF 8.83 INAQUEOUS ETHONAL, M.P. OF 239*-242* C. WITH DECOMPOSITION, (A)D24=-18*(C.1.0, N/10HCI), AN ELEMENTAL ANALYSIS OF C, 56:34; 8:51; N, 15:92, ANDA MOLECULAR WEIGHT OF ABOUT 1050+-50.
 2. The process for the preparationof the antibiotic Bu-1880, a basic peptide comprised of phenylalanine,leucine, Alpha , gamma -diaminobutyric acid and3-hydroxy-8-methyldecanoic acid in an approximate molar ratio of1:2:5:1, having the infrared spectrum of FIG. 1 and the followingphysical characteristics: pKa of 8.83 in aqueous ethanol, m.p. of239*-242* C. with decomposition, ( Alpha )D24 -18* (c. 1.0, N/10 HCl),an elemental analysis of C, 56.34; H, 8.51; N, 15.92, and a molecularweight of about 1050 + or - 50; which process comprises aerobicallyfermenting Bacillus circulans A.T.C.C. 21828 in acarbohydrate-proteinrich media for a period of about two to about threedays, at a pH of about 8 to about 8.5, at a temperature of about 28* C.,and then recovering the antibiotic by extraction with n-butanol.